In 1981, mouse embryonic stem cells have been first cultured in a test tube. In 1988, this method of in vitro culture has been combined with a gene targeting technique so as to first make a transformed mouse having a targeted gene. After that, the mouse gene targeting technique plays an important role to investigate genetic functions and to build up human disease models. Further, it drives life/medical sciences to develop (Smith A. G., Annu. Rev. Cell Dev. Biol., 17: 435-62, 2001). In contrast to the mouse case, human embryonic stem cells started to be cultured in vitro in 1998, 17 years later. Dr. Thompson has first established a human embryonic stem cell in University of Wisconsin (Thomson J. A., Science 282(5391): 1145-7, 1998). The human embryonic stem cell is more difficult to be cultured and manipulated than the mouse stem cell. Either, it is improper to be mass-cultured, even if necessary to develop therapeutic agents by performing a gene or in vitro manipulation. Therefore, it is required to complete a culture method that can proliferate stem cells effectively and control the quality easily.
The culture method popularly used is based upon the procedure Dr. Thompson established. Precisely, mouse embryonic fibroblasts are treated with mitomycin or irradiated in order to inhibit the cell growth. Then, the fibroblasts are inoculated to previously express extra-cellular substrates and cytokines embryonic stem cells need. The resulting cells are used for feeder cells, on which embryonic stem cells are inoculated. But, the optimal composition of culture medium of the stem cell is different from that of the feeder cell. To a basic medium of the embryonic stem cell, 20% serum replacement (Invitrogen Inc.) is added. In contrast, 20% fetal bovine serum (FBS) is added to a culture media of the feeder cell.
There are several disadvantages of stem cell culture. Because FBS is essential to survive fibroblasts, the fibroblast does not play a role of feeder cells when cultured with serum replacement instead of fetal bovine serum. In addition, this procedure reduces the cell survival of feeder cells to 5 to 7 days, since treating mitomycin or irradiating. In detail, stem cells should be sub-cultured before being confluent, even if they are first derived or delay a cell passage. Besides, feeder cells may not be safe due to mitomycin. Furthermore, stem cells are inevitably mixed with feeder cells when being sub-cultured, because 2 kinds of cells are cultivated on the same culture plate. This contamination causes a serious problem in case of clinical applications.
In order to settle above-mentioned problems, the present inventors have tried to co-culture stem cells with feeder cells by using a polymer membrane, in which 2 kinds of cells are cultivated in separate spaces while permeating essential substances selectively. As a result, the stem cell and the feeder cell can be cultured respectively under optimized conditions. Therefore, the present invention has been completed to provide the most optimal process for culturing stem cells successfully.